This application is designed to develop new therapeutic approaches for cartilage restoration, which in turn will reduce morbidity and mortality from degenerative joint disease. None of the current cartilage repair strategies has generated long lasting hyaline cartilage replacement tissue that meets functional demands placed upon this tissue in vivo. I hypothesize that human pluripotent stem cell (PSC) derived chondroprogenitor cells (ChPC) or the precursors of ChPC may have greater potential for use in regenerative medicine than adult articular chondrocytes and adult mesenchymal stem cells based on their chondrogenic commitment, lineage potential and proliferative ability. Further I propose that studies to identify and characterize the stages of human chondrogenesis are needed to insure successful generation and isolation of identical ChPC from PSC. Thus, the overall goal of the proposed study is to define the stages through which ChPC are generated from multipotent mesodermal ancestors, with the ultimate objective of producing ChPC with the highest potential for use in regenerative medicine. Our group has recently reported the earliest embryonic mesodermal progenitor (EMP) produced during human PSC differentiation. EMPs are ancestors of more lineage restricted mesenchymal progenitor cells (osteo- and chondrogenic), hematoendothelial and cardiovascular progenitors. I will use this novel EMP population to recapitulate early stages of embryonic mesenchyme generation, chondrogenesis and eventually, articular cartilage formation. I also carried out pilot immunophenotypical characterization of primary ChPC at early stages of human embryogenesis. In combination with in vitro studies of primary and induced PSC (iPSC)-derived ChPC populations, a xenograft animal model and in vivo bioluminescence imaging will be utilized in the following specific aims to study the potential of iPSC-derived ChPC for use in regenerative medicine. This application is submitted by Dr. Denis Evseenko, an investigator in the field of developmental and stem cell biology whose objective is to transition into an independent researcher. As part of his career development he will receive training in the new fields of cartilage biology and xenograft models of osteoarthritis. In addition this proposal outlines the course work through the NIH funded K30 program at UCLA and other training activities that will help Denis to increase his knowledge base and improve his grant writing skills and ability to execute translational clinical research. This application is strongly supported by the Orthopedic Research Hospital at UCLA. Dr John Adams who is a Vice Chair of Research Department of Orthopedic Surgery and Director of Orthopedic Hospital Research Center at UCLA will act as a primary mentor for this application. In summary, this application will serve not only to address immediate and long term scientific questions in the field of chondrogenesis, but also the career development of Dr Evseenko into a successful independent researcher.